Endocytosis--the process by which cells take up macromolecules-- plays an important role in both the normal metabolism of cells, as in the uptake of iron from endocytosed transferrin, and in disease processes, such as infection by enveloped viruses. Little, however, is known about the molecules that catalyze the endocytic process. The long term objective of this project is to define and characterize the molecular mechanisms by which cells take up macromolecules and deliver them to their appropriate destinations. Central to this delivery process is the cellular endosome, wherein the targeting decision is made for endocytosed materials. A cell-free system has been constructed, in which functions of the endosomal compartment have been reconstituted. The specific aim of this application is to identify, isolate, and characterize the components required for performing and regulating these endosomal functions. The endosomal functions reconstituted include the delivery of endocytosed materials into the endosomal compartment via vesicle fusion, and the sorting of those materials destined for delivery to lysosomes away from receptors destined to be recycled back to the cell surface. Both soluble cytosolic proteins and membrane-bound proteins have been associated with the fusion reaction that delivers materials to the endosome; these proteins, which include fusion-related proteins sensitive to N-ethylmaleimide, and regulatory proteins related to small molecular weight GTP-binding proteins, will be isolated, characterized, and their roles in delivery analyzed. The membrane components involved in endosomal function' ' will be identified, isolated, and analyzed for their role in promoting the sorting process associated with endosomal function, using antibodies directed against the exposed cytoplasmic tails of receptors known to transit this compartment during the endocytic process. These cell-free equivalents of the in vivo endosome will be examined for proteins, soluble or endosomally-associated, that catalyze the sorting function, as well as for membrane elements that constitute prelysosomal or recycling elements.